The regulation of proteolytic activity within the central nervous system (CNS) is essential in both normal and pathological processes. However, the precise role that proteolysis plays in the CNS is not well established. Recent work from many laboratories has indicated that the serine protease tissue-type plasminogen activator (tPA) is involved in a number of important processes both during development and in the adult brain. These include events associated with synaptic plasticity such as long term potentiation (LTP), motor learning and anxiety-like behavior, all processes which are though to involve synaptic remodeling. TPA has also been implicated in neuronal death following excitotoxic injury, seizure and stroke. The primary inhibitor of tPA within the CNS is the serine protease inhibitor (serpin) neuroserpin. The tissue distribution of neuroserpin indicates that it is predominantly expressed in neurons in areas where either tPA message or activity has also been localized. Neuroserpin is also found in areas known to have the highest susceptibility to ischemic injury, and neuroserpin is neuroprotective in stroke and seizure. Like tPA, neuroserpin has also been suggested to regulate anxiety-like behavior since both over-expression or complete deficiency of neuroserpin in mice leads to increased anxiety and neophobia. Thus, this proposal will focus on understanding the basic biology of neuroserpin and on its role in regulating tPA activity within the CNS. By using a combination of biochemical, molecular and genetic approaches both in vitro and in vivo, and building on previous studies of neuroserpin, and tPA, a number of important activities will be characterized and hypotheses tested. We will characterize the cell biology of neuroserpin and tPA within the CNS and test the hypothesis that tPA activity regulates neuroserpin release from neurons at the synapse. Sensitive assays will be used to localize neuroserpin and tPA and to monitor their trafficking and activity in primary neuronal cultures. We will also investigate the biochemical mechanisms of neuroserpin's regulation of tPA, and test the hypothesis that the inhibition of tPA by neuroserpin is regulated by pH. The role(s) of neuroserpin and tPA during normal and pathologic physiology such as anxiety and seizure will be examined in vivo, and we will test the hypothesis that neuroserpin and tPA regulate activity dependent changes in synaptic structure. Together these studies will give a better understanding of the roles of tPA and neuroserpin both normal and pathological processes, and may provide the basis for improved therapies for the treatment of CNS disorders.